The main goal of this proposal is to test the possibility that metabolic deficiencies can unmask mutation-related pathogenic roles of the cytoskeleton in digestive epithelia. Keratins, which represent the largest sub-class of intermediate filament (IF) proteins, form highly stable cytoskeletal structures that provide mechanical support and reinforce cellular resilience under stress. The cell-protective functions of keratins and other IF proteins have been extensively demonstrated in animal models, and are reflected in the vast number of tissue-specific human diseases linked to IF gene mutations. Heterozygous mutations in the gene that encodes keratin 8 (K8), the major IF protein in digestive epithelia, are susceptibility factors for acute and chronic liver diseases. The pathogenic effects of the known K8 variants, which exhibit structural filament changes, are only manifested upon cell injury, but the biological basis for this is poorly understood. The objective of this proposal is to identify specific molecular events behind the liver pathogenesis of K8 variants, with a particular focus on K8 aggregation in response to changes in energy metabolism. Our preliminary data from in silico analysis reveal that the known K8 variants cluster within regions of the protein that are most likely to form protofibrillar structures, which are generally considered to be cytotoxic. Furthermore, we have found that K8 solubility, oligomerization, and aggregation are regulated by site-specific K8 acetylation in response to changes in glucose and nicotinamide adenine dinucleotide (NAD) availability, as well as activity of the cytoplasmic NAD- dependent deacetylase sirtuin-2 (SIRT2). These findings raise the possibility that metabolic changes may trigger the pathologic manifestation of K8 mutations and lead us to hypothesize that mutation-induced keratin aggregation in digestive epithelia is a toxic event under the control of cellular metabolism. The proposed specific aims are designed to: (i) evaluate the influence of cellular metabolism on the aggregation properties of liver disease - associated keratin mutants; and, (ii) assess the effects of cellular metabolism on the toxicity of liver disease - associated keratin mutants. Upon completion of the proposed studies we expect to know if metabolic changes can tip the balance between mutant K8 filament disorganization and formation of toxic aggregates. This information, in turn, will serve as the basis for future studies aimed at targeting defective keratins by, for example, the use of pharmacological chaperones or kinetic stabilizers of the basic keratin filament subunits as potential novel approaches to treat keratin-related disorders.